TGF beta-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-beta (TGFbeta) enhanced cartilage repair and whether TGFbeta-induced fibrosis was blocked by local expression of the intracellular TGFbeta inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFbeta. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFbeta-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFbeta and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFbeta restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFbeta-induced an elevation in PG content in cartilage of the OA model. TGFbeta-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFbeta on cartilage. Adenoviral overexpression of TGFbeta stimulated repair of IL-1- and OA-damaged cartilage. TGFbeta-induced synovial fibrosis was blocked by locally inhibiting TGFbeta signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.     

TGF β-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-β (TGFβ) enhanced cartilage repair and whether TGFβ-induced fibrosis was blocked by local expression of the intracellular TGFβ inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFβ. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFβ-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFβ and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFβ restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFβ-induced an elevation in PG content in cartilage of the OA model. TGFβ-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFβ on cartilage. Adenoviral overexpression of TGFβ stimulated repair of IL-1- and OA-damaged cartilage. TGFβ-induced synovial fibrosis was blocked by locally inhibiting TGFβ signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.     

Effects of diacerein on biosynthesis activities of chondrocytes in culture

The maintenance of articular cartilage integrity requires a balance between anabolic and catabolic processes which are under the control of chondrocytes. These cells are living in an anaerobic environment and normally do not divide. They are responsible for the continuous maintenance of the cartilage extracellular matrix (ECM). Although multiple factors are involved in the dynamic homeostasis of cartilage, increases in cytokines such as interleukin-1 (IL-1) are associated with a decrease in synthesis and an increase in degradation of the proteoglycans and collagens. Conversely, growth factors such as transforming growth factor-beta (TGF-beta) stimulate chondrocyte synthesis of collagens and proteoglycans, and reduce the activity of IL-1 stimulated metalloproteases, thus opposing the inhibitory and catabolic effects of IL-1. By its capability to reduce IL-1 effects and to stimulate TGF-beta expression in cultured articular chondrocytes, diacerein could favour anabolic processes in the OA cartilage and, hence may contribute to delay the progression of the disease.     

Distinct pathways regulate facilitated glucose transport in human articular chondrocytes during anabolic and catabolic responses

Articular cartilage is an avascular, non-insulin-sensitive tissue that utilizes glucose as the main energy source, a precursor for glycosaminoglycan synthesis, and a regulator of gene expression. Facilitated glucose transport represents the first rate-limiting step in glucose metabolism. Previously, we demonstrated that glucose transport in chondrocytes is regulated by proinflammatory cytokines via upregulation of GLUT mRNA and protein expression. The objective of the present study was to determine differences in molecular mechanisms regulating glucose transport in chondrocytes stimulated with the anabolic transforming growth factor-beta1 (TGF-beta1) vs. the catabolic and proinflammatory cytokine IL-1beta. Both TGF-beta1 and IL-1beta accelerate glucose transport in chondrocytes. Although both IL-1beta and TGF-beta1 enhance glucose transport in chondrocytes to a similar magnitude, IL-1beta induces significantly higher levels of lactate. TGF-beta1-stimulated glucose transport is not associated with increased expression or membrane incorporation of GLUT1, -3, -6, -8, and -10 and depends on PKC and ERK activation. In contrast, IL-1beta-stimulated glucose transport is accompanied by increased expression and membrane incorporation of GLUT1 and -6 and depends upon activation of PKC and p38 MAP kinase. In conclusion, anabolic and catabolic stimuli regulate facilitated glucose transport in human articular chondrocytes via different effector and signaling mechanisms, and they have distinct effects on glycolysis.     

Aging, osteoarthritis and transforming growth factor-beta signaling in cartilage

Osteoarthritis is a common malady of the musculoskeletal system affecting the articular cartilage. The increased frequency of osteoarthritis with aging indicates the complex etiology of this disease, which includes pathophysiology and joint stability including biomechanics. The balance between anabolic morphogens and growth factors and catabolic cytokines is at the crux of the problem of osteoarthritis. One such signal is transforming growth factor-beta (TGF-beta). The impaired TGF-beta signaling has been identified as a culprit in old mice in a recent article in this journal. This commentary places this discovery in the context of anabolic and catabolic signals and articular cartilage homeostasis in the joint.     

IL-10 gene-deficient mice lack TGF-beta/Smad signaling and fail to inhibit proinflammatory gene expression in intestinal epithelial cells after the colonization with colitogenic Enterococcus faecalis

Nonpathogenic enteric bacterial species initiate and perpetuate experimental colitis in IL-10 gene-deficient mice (IL-10(-/-)). Bacteria-specific effects on the epithelium are difficult to dissect due to the complex nature of the gut microflora. We showed that IL-10(-/-) mice compared with wild-type mice fail to inhibit proinflammatory gene expression in native intestinal epithelial cells (IEC) after the colonization with colitogenic Gram-positive Enterococcus faecalis. Interestingly, proinflammatory gene expression was transient after 1 wk of E. faecalis monoassociation in IEC from wild-type mice, but persisted after 14 wk of bacterial colonization in IL-10(-/-) mice. Accordingly, wild-type IEC expressed phosphorylated NF-kappaB subunit RelA (p65) and phosphorylated Smad2 only at day 7 after bacterial colonization, whereas E. faecalis-monoassociated IL-10(-/-) mice triggered persistent RelA, but no Smad2 phosphorylation in IEC at days 3, 7, 14, and 28. Consistent with the induction of TLR2-mediated RelA phosphorylation and proinflammatory gene expression in E. faecalis-stimulated cell lines, TLR2 protein expression was absent after day 7 from E. faecalis-monoassociated wild-type mice, but persisted in IL-10(-/-) IEC. Of note, TGF-beta1-activated Smad signaling was associated with the loss of TLR2 protein expression and the inhibition of NF-kappaB-dependent gene expression in IEC lines. In conclusion, E. faecalis-monoassociated IL-10(-/-), but not wild-type mice lack protective TGF-beta/Smad signaling and fail to inhibit TLR2-mediated proinflammatory gene expression in the intestinal epithelium, suggesting a critical role for IL-10 and TGF-beta in maintaining normal epithelial cell homeostasis in the interplay with commensal enteric bacteria.     

Epigenetics of Osteoporosis: Critical Analysis of Epigenetic Epidemiology Studies

Osteoarthritis (OA) is an age-related disease with poorly understood pathogenesis. Recent studies have demonstrated that miRNA might play a key role in OA initiation and development. We reviewed recent publications and elucidated the connection between miRNA and OA cartilage anabolic and catabolic signals, including four signaling pathways: TGF-β/Smads and BMPs signaling, associated with cartilage anabolism; and MAPK and NF-KB signaling, associated with cartilage catabolism. We also explored the relationships with MMP, ADAMTS and NOS (NitricOxide Synthases) families, as well as with the catabolic cytokines IL-1 and TNF-α. The potential role of miRNAs in biological processes such as cartilage degeneration, chondrocyte proliferation, and differentiation is discussed. Collective evidence indicates that miRNAs play a critical role in cartilage degeneration. These findings will aid in understanding the molecular network that governs articular cartilage homeostasis and in to elucidate the role of miRNA in the pathogenesis of OA.     

Skeletal Characterization of Smurf2-Deficient Mice and In Vitro Analysis of Smurf2-Deficient Chondrocytes

Overexpression of Smad ubiquitin regulatory factor 2 (Smurf2) in chondrocytes was reported to cause spontaneous osteoarthritis (OA) in mice. However, it is unclear whether Smurf2 is involved in bone and cartilage homeostasis and if it is required for OA pathogenesis. Here we characterized age-related changes in the bone and articular cartilage of Smurf2-deficient (MT) mice by microCT and histology, and examined whether reduced Smurf2 expression affected the severity of OA upon surgical destabilization of the medial meniscus (DMM). Using immature articular chondrocytes (iMAC) from MT and wild-type (WT) mice, we also examined how Smurf2 deficiency affects chondrogenic and catabolic gene expressions and Smurf2 and Smurf1 proteins upon TGF-β3 or IL-1β treatment in culture. We found no differences in cortical, subchondral and trabecular bone between WT and MT in young (4 months) and old mice (16–24 months). The articular cartilage and age-related alterations between WT and MT were also similar. However, 2 months following DMM, young MT showed milder OA compared to WT (~70% vs ~30% normal or exhibiting only mild OA cartilage phenotype). The majority of the older WT and MT mice developed moderate/severe OA 2 months after DMM, but a higher subset of aged MT cartilage (27% vs. 9% WT) remained largely normal. Chondrogenic gene expression (Sox9, Col2, Acan) trended higher in MT iMACs than WT with/without TGF-β3 treatment. IL-1β treatment suppressed chondrgenic gene expression, but Sox9 expression in MT remained significantly higher than WT. Smurf2 protein in WT iMACs increased upon TGF-β3 treatment and decreased upon IL-1β treatment in a dose-dependent manner. Smurf1 protein elevated more in MT than WT upon TGF-β3 treatment, suggesting a potential, but very mild compensatory effect. Overall, our data support a role of Smurf2 in regulating OA development but suggest that inhibiting Smurf2 alone may not be sufficient to prevent or consistently mitigate post-traumatic OA across a broad age range.     

Chondroprotective effects of pulsed electromagnetic fields on human cartilage explants

This study investigated the effects of pulsed electromagnetic fields (PEMFs) on proteoglycan (PG) metabolism of human articular cartilage explants from patients with osteoarthritis (OA). Human cartilage explants, recovered from lateral and medial femoral condyles, were classified according to the International Cartilage Repair Society (ICRS) and graded based on Outerbridge scores. Explants cultured in the absence and presence of IL-1β were treated with PEMF (1.5 mT, 75 Hz) or IGF-I alone or in combination for 1 and 7 days. PG synthesis and release were determined. Results showed that explants derived from lateral and medial condyles scored OA grades I and III, respectively. In OA grade I explants, after 7 days exposure, PEMF and IGF-I significantly increased (35) S-sulfate incorporation 49% and 53%, respectively, compared to control, and counteracted the inhibitory effect of IL 1β (0.01 ng/ml). The combined exposure to PEMF and IGF-I was additive in all conditions. Similar results were obtained in OA grade III cartilage explants. In conclusion, PEMF and IGF-I augment cartilage explant anabolic activities, increase PG synthesis, and counteract the catabolic activity of IL-1β in OA grades I and III. We hypothesize that both IGF-I and PEMF have chondroprotective effects on human articular cartilage, particularly in early stages of OA.     

Human Osteoarthritic Cartilage Shows Reduced In Vivo Expression of IL-4, a Chondroprotective Cytokine that Differentially Modulates IL-1β-Stimulated Production of Chemokines and Matrix-Degrading Enzymes In Vitro

Background

In osteoarthritis (OA), an inflammatory environment is responsible for the imbalance between the anabolic and catabolic activity of chondrocytes and, thus, for articular cartilage derangement. This study was aimed at providing further insight into the impairment of the anabolic cytokine IL-4 and its receptors in human OA cartilage, as well as the potential ability of IL-4 to antagonize the catabolic phenotype induced by IL-1β.

Methodology/Principal Findings

The in vivo expression of IL-4 and IL-4 receptor subunits (IL-4R, IL-2Rγ, IL-13Rα1) was investigated on full thickness OA or normal knee cartilage. IL-4 expression was found to be significantly lower in OA, both in terms of the percentage of positive cells and the amount of signal per cell. IL-4 receptor type I and II were mostly expressed in mid-deep cartilage layers. No significant difference for each IL-4 receptor subunit was noted. IL-4 anti-inflammatory and anti-catabolic activity was assessed in vitro in the presence of IL-1β and/or IL-4 for 24 hours using differentiated high density primary OA chondrocyte also exhibiting the three IL-4 R subunits found in vivo. Chemokines, extracellular matrix degrading enzymes and their inhibitors were evaluated at mRNA (real time PCR) and protein (ELISA or western blot) levels. IL-4 did not affect IL-1β-induced mRNA expression of GRO-α/CXCL1, IL-8/CXCL8, ADAMTS-5, TIMP-1 or TIMP-3. Conversely, IL-4 significantly inhibited RANTES/CCL5, MIP-1α/CCL3, MIP-1β/CCL4, MMP-13 and ADAMTS-4. These results were confirmed at protein level for RANTES/CCL5 and MMP-13.

Conclusions/Significance

Our results indicate for the first time that OA cartilage has a significantly lower expression of IL-4. Furthermore, we found differences in the spectrum of biological effects of IL-4. The findings that IL-4 has the ability to hamper the IL-1β-induced release of both MMP-13 and CCL5/RANTES, both markers of OA chondrocytes, strongly indicates IL-4 as a pivotal anabolic cytokine in cartilage whose impairment impacts on OA pathogenesis.     

Gene expression profiling of serum- and interleukin-1 beta-stimulated primary human adult articular chondrocytes--a molecular analysis based on chondrocytes isolated from one donor

In order to understand the cellular disease mechanisms of osteoarthritic cartilage degeneration it is of primary importance to understand both the anabolic and the catabolic processes going on in parallel in the diseased tissue. In this study, we have applied cDNA-array technology (Clontech) to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one donor cultured under anabolic (serum) and catabolic (IL-1beta) conditions. Significant differences between the different in vitro cultures tested were detected. Overall, serum and IL-1beta significantly altered gene expression levels of 102 and 79 genes, respectively. IL-1beta stimulated the matrix metalloproteinases-1, -3, and -13 as well as members of its intracellular signaling cascade, whereas serum increased the expression of many cartilage matrix genes. Comparative gene expression analysis with previously published in vivo data (normal and osteoarthritic cartilage) showed significant differences of all in vitro stimulations compared to the changes detected in osteoarthritic cartilage in vivo. This investigation allowed us to characterize gene expression profiles of two classical anabolic and catabolic stimuli of human adult articular chondrocytes in vitro. No in vitro model appeared to be adequate to study overall gene expression alterations in osteoarthritic cartilage. Serum stimulated in vitro cultures largely reflected the results that were only consistent with the anabolic activation seen in osteoarthritic chondrocytes. In contrast, IL-1beta did not appear to be a good model for mimicking catabolic gene alterations in degenerating chondrocytes.     

Smad3 is essential for TGF-beta 1 to suppress IL-2 production and TCR-induced proliferation, but not IL-2-induced proliferation

Transforming growth factor-beta1 is essential to maintain T cell homeostasis, as illustrated by multiorgan inflammation in mice deficient in TGF-beta1 signaling. Despite the physiological importance, the mechanisms that TGF-beta1 uses to regulate T cell expansion remain poorly understood. TGF-beta1 signals through transmembrane receptor serine/threonine kinases to activate multiple intracellular effector molecules, including the cytosolic signaling transducers of the Smad protein family. We used Smad3(-/-) mice to investigate a role for Smad3 in IL-2 production and proliferation in T cells. Targeted disruption of Smad3 abrogated TGF-beta1-mediated inhibition of anti-CD3 plus anti-CD28-induced steady state IL-2 mRNA and IL-2 protein production. CFSE labeling demonstrated that TGF-beta1 inhibited entry of wild-type anti-CD3 plus anti-CD28-stimulated cells into cycle cell, and this inhibition was greatly attenuated in Smad3(-/-) T cells. In contrast, disruption of Smad3 did not affect TGF-beta1-mediated inhibition of IL-2-induced proliferation. These results demonstrate that TGF-beta1 signals through Smad3-dependent and -independent pathways to inhibit T cell proliferation. The inability of TGF-beta1 to inhibit TCR-induced proliferation of Smad3(-/-) T cells suggests that IL-2 is not the primary stimulus driving expansion of anti-CD3 plus anti-CD28-stimulated T cells. Thus, we establish that TGF-beta1 signals through multiple pathways to suppress T cell proliferation.     

Mechanisms for asporin function and regulation in articular cartilage

Osteoarthritis (OA), the most prevalent form of skeletal disease, represents a leading cause of disability following middle age. OA is characterized by the loss of articular cartilage; however, the details of its etiology and pathogenesis remain unclear. Recently, we demonstrated a genetic association between the cartilage extracellular matrix protein asporin and OA (Kizawa, H., Kou, I., Iida, A., Sudo, A., Miyamoto, Y., Fukuda, A., Mabuchi, A., Kotani, A., Kawakami, A., Yamamoto, S., Uchida, A., Nakamura, K., Notoya, K., Nakamura, Y., and Ikegawa, S. (2005) Nat. Genet. 37, 138-144). Furthermore, we showed that asporin binds to transforming growth factor-beta (TGF-beta), a key cytokine in OA pathogenesis, and inhibits TGF-beta-induced chondrogenesis. To date, functional data for asporin have come primarily from mouse cell culture models of developing cartilage rather than from human articular cartilage cells, in which OA occurs. Here, we describe mechanisms for asporin function and regulation in human articular cartilage. Asporin blocks chondrogenesis and inhibits TGF-beta1-induced expression of matrix genes and the resulting chondrocyte phenotypes. Small interfering RNA-mediated knockdown of asporin increases the expression of cartilage marker genes and TGF-beta1; in turn, TGF-beta1 stimulates asporin expression in articular cartilage cells, suggesting that asporin and TGF-beta1 form a regulatory feedback loop. Asporin inhibits TGF-beta/Smad signaling upstream of TGF-beta type I receptor activation in vivo by co-localizing with TGF-beta1 on the cell surface and blocking its interaction with the TGF-beta type II receptor. Our results provide a basis for elucidating the role of asporin in the molecular pathogenesis of OA.     

Smad-dependent cooperative regulation of interleukin 2 receptor alpha chain gene expression by T cell receptor and transforming growth factor-beta

The interleukin 2 receptor alpha chain (IL-2Ralpha) is a component of high affinity IL-2 receptors and thus critically regulates T cell growth and other lymphoid functions. Five positive regulatory regions together control lineage-restricted and activation-dependent IL-2Ralpha induction in response to antigen and IL-2. We now show that TGF-beta cooperates with T cell receptor (TCR) signaling to increase IL-2Ralpha gene expression. Moreover, we identify a sixth positive regulatory region that regulates IL-2Ralpha expression in cells treated with anti-CD3 + anti-CD28 as well as TGF-beta and show that this region contains binding sites for Smad3, AP-1, and cAMP-responsive element-binding protein/ATF proteins. The importance of Smad complexes is indicated by impaired IL-2Ralpha induction by TGF-beta in CD4+ T cells from both Smad3-/- and Smad4-/- mice. Thus, we have identified a novel positive regulatory region in the IL-2Ralpha gene that mediates TGF-beta-dependent induction of the gene. These findings have implications related to IL-2Ralpha expression on activated T cells and regulatory T cells.     

Xylosyltransferase-I regulates glycosaminoglycan synthesis during the pathogenic process of human osteoarthritis

Loss of glycosaminoglycan (GAG) chains of proteoglycans (PGs) is an early event of osteoarthritis (OA) resulting in cartilage degradation that has been previously demonstrated in both huma and experimental OA models. However, the mechanism of GAG loss and the role of xylosyltransferase-I (XT-I) that initiates GAG biosynthesis onto PG molecules in the pathogenic process of human OA are unknown. In this study, we have characterized XT-I expression and activity together with GAG synthesis in human OA cartilage obtained from different regions of the same joint, defined as "normal", "late-stage" or adjacent to "late-stage". The results showed that GAG synthesis and content increased in cartilage from areas flanking OA lesions compared to cartilage from macroscopically "normal" unaffected regions, while decreased in "late-stage" OA cartilage lesions. This increase in anabolic state was associated with a marked upregulation of XT-I expression and activity in cartilage "next to lesion" while a decrease in the "late-stage" OA cartilage. Importantly, XT-I inhibition by shRNA or forced-expression with a pCMV-XT-I construct correlated with the modulation of GAG anabolism in human cartilage explants. The observation that XT-I gene expression was down-regulated by IL-1β and up-regulated by TGF-β1 indicates that these cytokines may play a role in regulating GAG content in human OA. Noteworthy, expression of IL-1β receptor (IL-1R1) was down-regulated whereas that of TGF-β1 was up-regulated in early OA cartilage. Theses observations may account for upregulation of XT-I and sustained GAG synthesis prior to the development of cartilage lesions during the pathogenic process of OA.     

TGF-beta and Smad3 signaling link inflammation to chronic fibrogenesis

Transient adenovirus-mediated gene transfer of IL-1beta (AdIL-1beta), a proinflammatory cytokine, induces marked inflammation and severe and progressive fibrosis in rat lungs. This is associated with an increase in TGF-beta1 concentration in bronchoalveolar lavage (BAL) fluid. TGF-beta1 is a key cytokine in the process of fibrogenesis, using intracellular signaling pathways involving Smad2 and Smad3. In this study we investigate whether inflammation induced by IL-1beta is able to independently induce lung fibrosis in mice deficient in the Smad3 gene. Seven days after AdIL-1beta administration, similar levels of IL-1beta transgene are seen in BAL in both wild-type (WT) and knockout (KO) mice, and BAL cell profiles demonstrated a similar marked neutrophilic inflammation. Phospho-Smad2 staining was positive in areas of inflammation in both WT and KO mice at day 7. By day 35 after transient IL-1beta expression, WT mice showed marked fibrosis in peribronchial areas, quantified by picrosirius red staining and morphometry. However, there was no evidence of fibrosis or collagen accumulation in IL-1beta-treated KO mice, and peribronchial areas were not different from KO mice treated with the control adenovector. TGF-beta1 and phospho-Smad2 were strongly positive at day 35 in fibrotic areas observed in WT mice, but no such staining was detectable in KO mice. The IL-1beta-induced chronic fibrotic response in mouse lungs is dependent on Smad3. KO and WT animals demonstrated a similar inflammatory response to overexpression of IL-1beta indicating that inflammation must link to the Smad3 pathway, likely through TGF-beta, to induce progressive fibrosis.